ErmB导致大环内酯耐药的机制研究

发布时间：2018-07-02 08:26

  本文选题：大环内酯 + rRNA　； 参考：《华中农业大学》2017年硕士论文

【摘要】：大环内酯类抗生素在临床广泛应用,主要用于治疗革兰氏阳性菌引起的感染,而细菌对大环内酯耐药现象日益严重。甲基化酶erm基因是导致大环内酯高水平耐药的重要机制之一。文献认为erm催化23S rRNA 2058位腺嘌呤核苷酸的修饰,使其单甲基化或二甲基化。甲基化后的空间结构使细菌与大环内酯的结合位点被隐藏,阻断了大环内酯等抗生素与细菌核糖体之间的结合,从而导致大环内酯、林可酰胺及链霉杀阳菌素耐药。但是erm甲基化作用机制及其对细菌耐药程度的贡献在国际国内尚未见报道。本实验室于临床发病雏鸭脑和脾脏中分离的解没食子酸链球菌巴氏亚种(S.gallolyticus subsp.pasteurianus)中发现耐药基因ermB和ermT,且这两种erm基因介导这些分离株大环内酯高水平耐药。此外,我们还发现不同分离株中ermB单核苷酸多态性(Single Nucleotide Polymorphism,SNP)引起ermB突变,且其大环内酯MIC存在差异。为了探讨erm甲基化作用机制及其对大环内酯耐药的贡献,本研究优化了ermB和ermT表达体系,选取了ermB进行研究,并基于生物信息学分析及临床分离株中的SNP构建了32种ermB突变体,首次利用微量稀释法对这32种突变体进行筛选,选取了六个突变体,分别检测了它们对23S rRNA的甲基化活性和对大环内酯及林可酰胺的最小抑菌浓度(MIC)。研究结果如下:1、erm B、ermT甲基化酶表达载体的构建与纯化及23S rRNA的分离纯化将ermT耐药基因分别构建到pET28a、pET15b载体上,ermB耐药基因分别构建到pET21b、pET28a载体上,比较蛋白分离纯化结果,选择ermB、ermT构建至pET28a载体的克隆,利用亲和层析纯化出ErmB、ErmT蛋白、MBP-MS2蛋白以及S.gallolyticus subsp.pasteurianus 23S r RNA。2、erm B、ermT的甲基化活性比较用3H标记S-腺苷甲硫氨酸为甲基供体,23S r RNA为底物,对ErmB、ErmT进行酶促动力学试验。两个蛋白的甲基化活性通过液闪仪检测转移到23S rRNA上3H的放射性强度完成。试验结果表明,ErmB和ErmT均可以甲基化修饰S.gallolyticus subsp.pasteurianus 23S r RNA,但ermB甲基化活性要低于ermT。3、erm B突变体构建结合实验室前期解没食子酸链球菌分离株中ermB的SNP,我们选取ermB进行研究。运用生物信息学方法分析erm及ermB同源序列,对ermB保守区域部分氨基酸残基进行突变,构建了32个突变体。4、erm B、ermB突变体MIC值检测粗筛对ermB及其32个突变体进行MIC值检测。结果表明,与野生型ermB的MIC值相比,32株突变体中对红霉素MIC降低的突变体包括:突变菌株40(K40A)、69-2(E69D)、83-1(D83A)、139(R139A);对林可酰胺MIC降低的突变体包括:突变菌株40(K40A)、69-2(E69D)、116-1(E116A)、139(R139A);对克拉霉素MIC降低的突变体包括:突变菌株58-1(E58A)、58-2(E58D)、60-2(E69D)、83-1(D83A)、139(R139A)、165(K165A)。5、微量稀释法测定6株突变体的MIC值通过ermB、ermB突变体MIC值检测粗筛选择6株ermB突变体进行精确MIC值检测,检测结果显示,突变菌株83-1(D83A)对红霉素、克拉霉素、林可酰胺的MIC值分别为:512μg/mL、64μg/mL、512μg/mL;突变菌株139139(R139A)对三种抗生素的MIC值分别为:512μg/mL、64μg/mL、128μg/mL;突变菌株165165(K165A)对三种抗生素的MIC值分别为:1024μg/mL、128μg/mL、128μg/mL;突变菌株4040(K40A)对三种抗生素的MIC值分别为512μg/mL、128μg/mL、256μg/mL;突变菌株37(G37A)对三种抗生素的MIC值分别为:1024μg/mL、256μg/mL、512μg/mL;突变菌株39(G39A)对三种抗生素的MIC值分别为:1024μg/mL、512μg/mL、1024μg/mL。6、erm B及erm B突变体甲基化活性比较用3H标记S-腺苷甲硫氨酸为甲基供体,23S rRNA为底物,对野生型及突变体ErmB的甲基化活性进行检测。试验结果表明,突变体37(G37A)、39(G39A)酶活性高于野生型ermB甲基化酶活性,突变体40(K40A)、83-1(D83A)、139(R139A)、165(K165A)酶活性小于野生型ermB甲基化酶活性。7、erm B甲基化酶生物进化树构建利用Mrbays3.1.2生物信息学软件对ermB不同物种进行同源性分析,构建了ermB甲基化酶的系统发育树。结论:ermB、ermT基因构建到pET28a载体上蛋白纯化效果较好。构建的32个ermB突变体与野生型ermB的MIC值相比,对红霉素MIC降低的突变体包括:突变菌株40(K40A)、69-2(E69D)、83-1(D83A)、139(R139A);对林可酰胺MIC降低的突变体包括:突变菌株40(K40A)、69-2(E69D)、116-1(E116A)、139(R139A);对克拉霉素MIC降低的突变体包括:突变菌株58-1(E58A)、58-2(E58D)、60-2(E69D)、83-1(D83A)、139(R139A)、165(K165A)。突变菌株37(G37A)、39(G39A)获得的酶活性大于野生型ermB,突变菌株40(K40A)、83-1(D83A)、139(R139A)、165(K165A)获得的酶活性小于野生型ermB,与MIC值变化趋势相符。以上结果说明,氨基酸K40、D83、R139及K165对ermB甲基化活性具有重要作用,这些氨基酸残基进一步影响大环内酯耐药。本研究为揭示大环内酯耐药机制提供了信息,首次建立了MIC值与erm酶活性的关系,为解决核糖体甲基化产生的抗生素耐药问题提供了新的方向。
[Abstract]:Macrolide antibiotics are widely used in clinical application, mainly for the treatment of gram positive bacteria infection, and bacterial resistance to macrolide is increasingly serious. Methylation enzyme ERM gene is one of the important mechanisms leading to the high level of macrolide resistance. It is believed that ERM catalyzes the modification of 23S rRNA 2058 adenine nucleotides to make them Monomethylation or two methylation. The spatial structure of methylation causes the binding sites of bacteria and macrolides to be hidden, blocking the combination of macrolides and bacterial ribosomes, leading to the resistance to macrolides, linacamide and streptomycin. But the mechanism of ERM methylation and the tribute to the degree of resistance to bacteria In the laboratory, the drug resistant gene ermB and ermT were found in S.gallolyticus subsp.pasteurianus isolated from the brain and spleen of ducklings, and these two ERM genes mediate the high level resistance of these isolates, and we also found different isolates. The ermB single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) in the plant causes ermB mutation and its macrolide MIC is different. In order to explore the mechanism of ERM methylation and its contribution to the resistance to macrolide, this study optimized the ermB and ermT expression system, selected ermB to study, and based on bioinformatics analysis and clinical practice. 32 ermB mutants were constructed by SNP in the isolated strain. The 32 mutants were screened by microdilution method for the first time and six mutants were selected. The methylation activity of 23S rRNA and the minimum inhibitory concentration (MIC) for macrolide and linlacamide were detected respectively. The results were as follows: 1, ERM B, ermT methylation enzyme expression vector Construction and purification and separation and purification of 23S rRNA were constructed to pET28a, pET15b vector, and ermB resistant genes were constructed on pET21b, pET28a vector, and compared with the results of protein separation and purification. ErmB, ermT were constructed to the clone of pET28a carrier, and ErmB, protein, protein, and purified protein were purified by affinity chromatography. The methylation activity of yticus subsp.pasteurianus 23S R RNA.2, ERM B, ermT was compared with 3H labeled S- adenosine methionine as the methyl donor and 23S r as the substrate, and the enzyme catalyzed kinetic test. The methylation activity of the two proteins was transferred to the radioactive intensity by the liquid flicker detection. S.gallolyticus subsp.pasteurianus 23S R RNA can be modified by methylation and ErmT, but ermB methylation activity is lower than ermT.3 and ERM B mutants are constructed in conjunction with ermB SNP of Streptococcus gallate isolates in the early laboratory. Partial amino acid residues in the region were mutated, and 32 mutant.4, ERM B, ermB mutant MIC values were detected for ermB and 32 mutants to be detected by MIC values. The results showed that, compared with the MIC value of the wild type ermB, the mutants of erythromycin MIC decreased in 32 mutants include: Mutant strain 40 (K40A), 69-2 (E69D), 83-1 (139), 139) 139A); mutants that reduce the MIC of linclamides include mutant strain 40 (K40A), 69-2 (E69D), 116-1 (E116A), 139 (R139A), and mutants of clarithromycin MIC, including mutant 58-1 (E58A), 58-2 (E58D), 60-2 (E69D), 83-1 (D83A), 139 (R139A), 165 The results showed that the MIC values of the mutant strain 83-1 (D83A) to erythromycin, clarithromycin and linclamides were 512 mu g/mL, 64 mu g/mL, 512 micron g/mL, and the MIC values of three mutant strains 139139 (R139A) to three antibiotics were 512 mu g/mL, 64 mu g/mL, 128 mu g/mL, and mutant isolates 165165. (K165A) the MIC values for three kinds of antibiotics were 1024 mu g/mL, 128 mu g/mL, 128 mu g/mL, and the MIC values of the mutant 4040 (K40A) to three antibiotics were 512 mu g/mL, 128 micron and 256 micron g/mL respectively. The MIC values of the mutant 37 (G37A) to three antibiotics were respectively 1024 mu, 256, and 512 mu; The methylation activity of g/mL, ERM B and ERM B mutants was compared with 1024 mu, 512 mu, 1024 mu g/mL.6, ERM B and ERM B, and 3H labeled S- adenosine methionine as the methyl donor and 23S rRNA as the substrate to detect the methylation activity of the wild type and mutant ErmB. The experimental results showed that the mutant 37 was higher than the wild type methylation enzyme activity. Sex, the mutant 40 (K40A), 83-1 (D83A), 139 (R139A), 165 (K165A) enzyme activity is less than the wild type ermB methylation enzyme activity.7, ERM B methylation enzyme biological evolution tree construction uses the Mrbays3.1.2 bioinformatics software to analyze the homology of ermB species, and constructs the phylogenetic tree of ermB methylation enzyme. The protein purification effect on the 8A carrier is better. Compared with the MIC value of the wild type ermB, the 32 ermB mutants for erythromycin MIC include mutant strain 40 (K40A), 69-2 (E69D), 83-1 (D83A), 139 (R139A), and mutant strain 40 (K40A), 69-2 (E69D), 116-1 (139), 139 (139)); Mutants reduced by mycophenin MIC, including mutant strain 58-1 (E58A), 58-2 (E58D), 60-2 (E69D), 83-1 (D83A), 139 (R139A), 165 (K165A), mutant strain 37 (G37A), and 39 (G39A) were more active than wild type ermB, mutant strain 40 (K40A), 83-1 (D83A), 139, and 165 (139), which were less than wild type, and were consistent with the trend of variation. These results suggest that amino acid K40, D83, R139 and K165 have an important role in the ermB methylation activity. These amino acid residues further affect the resistance of macrolides. This study provides information for revealing the mechanism of macrolide resistance and the first establishment of the relationship between the MIC and the activity of the ERM enzyme, in order to solve the antibiotic resistance produced by the ribosome methylation. The problem provides a new direction.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.7

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